Through the collaborative and individual efforts of four laboratories we propose to carry out a coordinated analysis of the functions of a newly discovered family of proteins named MAGUKs (Membrane-Associated GUanylate Kinase Homologs) that appear to have important structural and signalling roles at the cell membrane in both invertebrates and vertebrates. These proteins all contain a domain with homology to guanylate kinase (GUK), an SH3 domain and one or three additional domains of homology (DHRs) for which the function is unknown. In project #1, molecular genetic methods will be used to investigate the MAGUK DlgA, which is a component of Drosophila septate junctions and is required for maintaining septate junction structure, cell shape, polarity and proliferation control. In Project #2, biochemical methods will be used to identify proteins that interact with the MAGUKs, to map the binding sites and to investigate how binding to other proteins affects function. This project will focus on p55, which was identified from human erythrocyte membranes, and Hdlg, which was identified from lymphocytes; both proteins have been shown to bind to other proteins that link the cytoskeleton to the membrane and stabilize cell shape. Project #3 concerns the possible signalling functions of Z0-1 and Z0-2, MAGUKs localized in mammalian tight junctions required for maintaining tissue structure, transepithelial permeability barriers, and cell polarity. Project #4 will be a molecular genetic analysis of the MAGUK LIN-2 of Caenorhabditis, which contains an additional N-terminal domain homologous to calcium/calmodulin-dependent protein kinases; Project #1 also includes a genetic analysis of the corresponding Drosophila gene. Using a combination of molecular, genetic and cell biological approaches, we will determine which domains of the MAGUKs are responsible for controlling their subcellular localization, what proteins associate with them, and how they interact with known signal transduction pathways. Biochemical assays will be used to determine the catalytic and/or regulatory role of the GUK domains, and to test whether these proteins act as substrates for tyrosine kinases. The participating laboratories will bring together experimental systems with unique advantages, and they will contribute expertise in biochemistry, genetics and cell biology, to define the functions of a remarkable new family of proteins that have important roles in the control of cell structure, differentiation and proliferation.